Dynabeads M-450 Epoxy

Intended Use

Dynabeads™ M-450 Epoxy coupled with antibodies or other ligands provide a versatile tool for isolation of both cells and non-cell targets (e.g. proteins and other biomolecules). Their size makes them particularly suitable for stimulation and expansion of e.g. T cells (13, 14). Dynabeads M-450 Epoxy may, without coupling of ligands, be used to deplete phagocytic cells (12).

Note:  Dynabeads ClinExVivo™ Epoxy (Cat. no. 402.01D) is available for ex vivo separation of human cells for cell-based clinical research.

Principle of Coupling

Dynabeads M-450 Epoxy are hydrophobic and covered with surface epoxy groups. The epoxide chemistry immobilises ligands containing amino, thiol and hydroxyl functional groups. No further activation is necessary an  irreversible binding of the ligands to the epoxy groups is achieved over a wide temperature range and at a neutral to alkaline pH.

Principle of Isolation

Once coupled with the specific ligand, the Dynabeads are mixed with the sample in a tube. The Dynabeads will bind to the target during a short incubation, and then the bead-bound target is separated by a magnet.

• Positive isolation – discard the supernatant and use the bead-bound target for downstream applications.
• Depletion – discard the bead-bound target and use the remaining, untouched sample for downstream applications.

Description of Materials

Dynabeads M-450 Epoxy are uniform, superparamagnetic polystyrene beads (4.5 μm diameter) with a surface suitable for physical and chemical binding of antibodies and other biomolecules.


Materials Supplied

5 ml Dynabeads M-450 Epoxy 4 x 10⁸ beads/ml supplied in distilled water.

Additional Materials Required

• Magnet: (Dynal MPC™) - MPC-S for 20 μl to 2 ml samples, MPC-L for 1-15 ml samples and MPC-50 for 15-50 ml samples.

• Mixer allowing both tilting and rotation.
• Buffer 1: 0.1 M sodium phosphate buffer, pH 7.4-8.0 or 0.1 M sodium borate buffer, pH 7.6-9.5.
• Buffer 2: phosphate buffered saline (PBS) with 0.1% bovine serum albumin (BSA), pH 7.4.
• Optional: Sodium azide (NaN₃) as preservative.
• Specific ligands.

Dynabeads Washing Procedure

Dynabeads should be washed before use.

1. Resuspend the Dynabeads in the vial.


2. Transfer the desired volume of Dynabeads to a tube.


3. Add the same volume of Buffer 1, or at least 1 ml, and mix.


4. Place the tube in a magnet for 1 min and discard the supernatant.


5. Remove the tube from the magnet and resuspend the washed Dynabeads in the same volume of Buffer 1 as the initial volume of Dynabeads.
   

Critical Steps for Coupling of Ligands to Dynabeads
 
Buffers

• Sugars and stabilisers may interfere with binding and should be removed from the ligand preparation.
• Coupling buffers should not contain any reactive groups (amines, thiols and hydroxyls) e.g. tris, glycine or proteins.
• The pH and the ionic strength of the coupling buffer can be varied, but generally the coupling reaction is carried out in 0.1 M phosphate buffer at pH 7.4-8.0. Epoxy groups are more reactive at higher pH, therefore 0.1 M borate buffer pH 7.6-9.5 can be used depending on the ligand stability. Buffers with higher ionic strength stimulate hydrophobic interactions, which facilitate the coupling efficiency.

Ligand and Dynabeads concentration

• For cell separation purposes couple with 3-5 μg purified ligand per 25 μl (1 x 10⁷) Dynabeads. Optimize the amount of ligand.
• The optimal Dynabeads concentration during coupling is 4-8 x 10⁸ Dynabeads per ml.

Coupling conditions

• Generally, temperatures from 18-37°C and incubation times from 16-24 hours are recommended. The upper temperature is limited by ligand stability. The rate of covalent coupling of the ligand to the Dynabeads increases with temperature.
• Temperature-labile ligands may be coupled at 2-8°C with prolonged incubation.

Blocking

• Adding a blocking protein such as 0.01-0.5% w/v BSA to the coupling solution may increase functionality of the coupled antibodies in cell isolation protocols. Blocking protein is generally added to coupling solution after 0-30 min. Both the incubation time before blocking and blocking protein concentration should be optimized.
• For protein isolation applications, blocking may not be required.

Coupling of Ligands to Dynabeads

The following procedure is a suggested, general procedure for antibody coupling to Dynabeads. It can be scaled-up and optimized.

• Use approximately 5 μg antibody per 25 μl (1 x 10⁷) Dynabeads.
• This procedure describes coupling to 1 ml (4 x 10⁸) Dynabeads.

1. Transfer 1 ml of washed Dynabeads to a tube.


2. Place the tube in a magnet for 1 min and discard the supernatant. Remove the tube from the magnet.


3. Resuspend Dynabeads in Buffer 1 (1 ml minus antibody volume) and add 200 μg antibody during mixing to reach a total coupling volume of 1 ml.


4. Incubate for 15 min and then add BSA to 0.1% w/v.


5. Incubate for 16-24 hours at 18-25°C (RT) with gentle tilting and rotation.


6. Place the tube in a magnet for 1 min and discard the supernatant.


7. Add 1 ml Buffer 2, mix and incubate for 5 min with gentle tilting and rotation.


8. Repeat step 6-7 twice.


9. Remove the tube from the magnet and resuspend the Dynabeads in 1 ml Buffer 2 (to obtain 4 x 10⁸ beads/ml).


10. For cell isolation, proceed to Critical Steps for Cell Isolation

Sample Preparation

Cells can be directly isolated from any sample such as whole blood (1, 3), bone marrow (7), mononuclear cell suspensions (MNC) or tissue digests. Please visit www.lifetechnologies.com/samplepreparation for a list of recommended sample preparation procedures.

Critical Steps for Cell Isolation

• Use a mixer that provides tilting and rotation of the tubes to ensure Dynabeads do not settle at the bottom of the tube.
• When incubating Dynabeads and cells, the incubation temperature must be 2 - 8°C to reduce phagocytic activity and other metabolic processes.
• Never use less than 25 μl (1 x 10⁷) Dynabeads per ml cell sample and at least 4 Dynabeads per target cell.

Table 1: Volume of Dynabeads added per ml of cell sample. The volumes can be scaled up as required.

* If the concentration of cells is increased or the target cell concentration exceeds 2.5 x 10⁶, the Dynabeads volume must be increased accordingly. Cell concentration can be up to 1 x 10⁸ cells per ml.


Cell Isolation

Wash coupled Dynabeads before use to remove any soluble ligand. Use the Dynabeads Washing Protocol at beginning of the Protocols section, replacing Buffer 1 with Buffer 2.

1. Add Dynabeads to the prepared sample according to table 1.


2. Incubate for 20 min (positive isolation) or 30 min (depletion) at 2 - 8°C with gentle tilting and rotation.


3. Optional: Double the volume with Buffer 2 to limit trapping of unbound cells.


4. Place the tube in a magnet for 2 min.


5. Depletion: Transfer the supernatant containing the unbound cells to a fresh tube for further experiments.


6. Positive isolation: Discard the supernatant and gently wash the bead-bound cells 4 times, using the following procedure:    i) Add 1 ml Buffer 2 per 1 x 10⁷ Dynabeads.  ii) Place the tube in the magnet for 1 min and discard the supernatant.


7. Resuspend the cells in buffer/medium for downstream application.

Antibody Selection

The choice of primary antibody is the most important factor for successful cell isolation. Note that some antibodies may show reduced antigen-binding efficiency when coated directly onto beads, even if the antibody shows good results in other immunological assays. See section below for recommendation of our secondary coated products.

Spacer and Ligand Orientation

The efficiency of antibody-antigen binding can be increased for some applications by using a spacer molecule e.g. a secondary antibody coupled to the Dynabeads prior to coupling of the primary antibody. Invitrogen Dynal offers several secondary coated Dynabeads for cell isolation. 

Storage of Coupled Dynabeads

• Store coupled Dynabeads in Buffer 2 at 2-8°C in liquid suspension. Coupled Dynabeads may typically be stored for months or even years. Stability of coupled Dynabeads depends on ligand stability and must be determined separately.
• A final concentration of 0.02% NaN₃ can be added as a preservative to the storage buffer. The preservative must be removed by washing prior to cell isolation

Isolation and Depletion of Target Cells

• Remove density gradient media (e.g. Ficoll): Wash cells prior to adding antibodies or Dynabeads.
• Remove soluble factors in serum: Serum may contain soluble factors (e.g. antibodies or cell surface antigens), which can interfere with the cell isolation protocol. Washing the cells once may reduce this interference.

Detachment of Cells from Dynabeads

Some cells exhibit high turnover of expressed antigens and will detach from Dynabeads during 6-24 hours of incubation under culture conditions (37°C).

1. Vartdal F et al (1986) HLA Class I and II typing using cells positively selected from blood by immunomagnetic isolation: A fast and reliable technique. Tissue Antigens 28:301-312.


2. Egeland T et al (1991) Myeloid differentiation of purified CD34⁺ cells after stimulation with recombinant Human Granulocyte-Monocyte Colony-Stimulating Factor (CSF), Granulocyte-CSF, Monocyte-CSF, and Interleukin-3. Blood 78(12):3192-3199


3. George F et al (1992) Rapid isolation of human endothelial cells from whole blood using S-Endo1 monoclonal antibody coupled to immuno-magnetic beads: Demonstration of endothelial injury after angioplasty. Thrombosis and Haemostasis 67(1):147-153


4. Naume B et al (1992) A comparative study of IL-12 (Cytotoxic Lymphocyte Maturation Factor), IL-2-, and IL-7-induced effects on immunomagnetically purified CD56 NK cells. J. Immunol. 148(8):2429-2436


5. Hansel TT et al (1989) Purification of human blood eosinophils by negative selection using immunomagnetic beads. J Immunol. Meth. 122: 97-103


6. Manyonda IT et al (1992) A critical evaluation of the magnetic cell sorter and its use in the positive and negative selection of CD45RO⁺ cells. J Immunol. Meth. 149:1-10


7. Wang MY et al (1992) An effective immunomagnetic method for bone marrow purging in Tcell malignancies. Bone Marrow Transplant 9:319-323


8. Alfonso M et al (1995) Antigen-specific primary immune response of human B-lymphocytes after in vitro immunization with GM3 ganglioside. Human Antibody Hybridomas 6:102-112


9. Barcellini W et al (1992) Enrichment of IgG anti-DNA-producing lymphoblastoid cell lines by antigen-coated immunomagnetic beads. Clin. Immunol. Immunopathol. 65:39-44


10. Hougs L et al (1993) Rapid analysis of rearranged kappa light chain genes of circulating polysaccharide- specific B lymphocytes by means of immunomagnetic beads and the polymerase chain reaction. Exp. Clin. Immunogenet  10: 141-151


11. Schweitzer CM et al (1995) Isolation and culture of human bone marrow endothelial cells. Exp. Hematol. 23:41-48


12. Thompson JA et al (2003) A phase I trial of CD3/CD28-activated T cells (Xcellerated T cells) and Interleukin-2 in patients with metastatic renal cell carcinoma. Clin. Cancer Res. 9:3562-3570


13. Kovacs B et al (2002) Human CD8+ T cells do not require the polarization of lipid rafts for activation and proliferation. PNAS 99(23):15006-15011


14. Maus MV et al (2004) Extensive replicative capacity of human central memory T cells. J. Immunol. 172:6675-6683


15. Xiaobin L et al (2004) Antisense-mediated inhibition of Human Immunodeficiency Virus (HIV) replication by use of an HIV Type 1-based vector results in severely attenuated mutants incapable of developing resistance. J. Virol. 78(13):7079-7088


16. Florence L et al (2002) A proteomic view of the Plasmodium falciparum life cycle. Nature 419: 520-526


17. Morton HC et al (2004) Characterization of the ligand binding site of the bovine IgA Fc receptor (bFcaR). J. Biol. Chem. 279(52):54018-54022


18. Perez OD et al (2004) LFA-1 signaling through p44/42 is coupled to perforin degranulation in CD56⁺CD8⁺ natural killer cells. Blood 104(4): 1083-1093


19. Rharbaoui F et al (2005) Characterization of a B220⁺ lymphoid cell subpopulation with immune modulatory functions in nasal-associated lymphoid tissues. J. Immunol. 174:1317-1324